The long term objective of our work is to better understand the pathogenesis of Graves'ophthalmopathy (GO) in order to aid in the development of new approaches to treatment or prevention of this disease. The goal of the laboratory investigations in this proposal is to determine mechanisms involved in the stimulation of adipogenesis by thyrotropin receptor (TSHR)-directed autoantibodies (TRAb) in GO orbital preadipocytes. The translational goal is to determine whether rituximab, an agent that blocks the activation and differentiation of B cells, confers therapeutic benefit in a pilot study of patients with GO. We have shown that adipogenesis is enhanced in orbital tissues from GO patients, and that TRAb act to stimulate fat cell development in GO orbital preadipocytes. Further, we have demonstrated elevated expression of soluble frizzled-related protein-1 (sFRP-1) in patients'orbital tissue specimens, and present evidence that TRAb enhance expression of this protein in their orbital preadipocytes. sFRP-1 is an inhibitor of Wnt signaling that acts in preadipocytes to reverse Wnt-induced inhibition of adipogenesis, thereby enhancing fat cell development. Analogous to TRAb activating thyroidal TSHR and stimulating over-production of thyroid hormone in Graves'disease, we postulate that these autoantibodies activate/TSHR on orbital preadipocytes to stimulate adipogenesis in GO. The 3 specific aims of this proposal are to test the hypotheses that: 1) TRAb-induced adipogenesis is mediated via inhibition of Wnt signaling, and that IgG from GO patients function similarly;2) GO orbital preadipocytes differ from preadipocytes from other sites in their responses to TRAb;and 3) Rituximab is effective in the treatment of patients with severe, active GO. We will enroll 30 patients in a randomized, prospective, double-blind clinical trial to determine effects of this agent on clinical activity score, specific quantitative ocular parameters, and health-related quality of life. We believe that our program represents a novel, integrated, translational approach to the study of GO. Our studies will yield information concerning mechanisms involved in disease development, and will determine the effectiveness of a novel approach to therapy.